High-throughput screening method and apparatus

ABSTRACT

High-throughout screening method and apparatus are described. The method includes placing cells on a substrate defining a plurality of discrete microwells, at a well density of greater than about 100/cm 2 , with the number of sells in each well being less that about 1000, and where the cells in each well have been exposed to a selected agent. The change in conductance in each well is determined by applying a low-voltage, AC signal across a pair of electrodes placed in that well, and synchronously measuring the conductance across the electrodes, to monitor the level of growth or metabolic activity of cells contained in each well. Also disclosed is an apparatus for carrying out the screening method.

This application claims benefit of Provision Application No. 60/021,074Jun. 27, 1996.

FIELD OF THE INVENTION

The present invention relates to high throughput screening (HTS)methods, e.g., for detecting the effect of a given compound or treatmenton cell metabolic activity, and apparatus for performing such screening.

BACKGROUND OF THE INVENTION

With the advent of combinatorial library methods for generating largelibraries of compounds, there has been a growing interest inhigh-throughput screening (HTS) methods for screening such libraries.

The most widely used HTS screening method involves competitive ornon-competitive binding of library compounds to a selected targetprotein, such as an antibody or receptor. Thus, for example, to select alibrary compound capable of blocking the binding of a selected agonistto a receptor protein, the screening method could assay for the abilityof library compounds to displace radio-labeled agonist from the targetprotein.

Although such binding assays can be used to rapidly screen large numbersof compounds for a selected binding activity, the assay itself may havelimited relevance to the actual biological activity of the compound invivo, e.g., its ability to interact with and affect the metabolicbehavior of a target cell.

It would therefore be useful to provide high throughput screeningmethods capable of testing the effects of large numbers of librarycompounds on target cells of interest.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, high throughput screeningapparatus, e.g., for screening the effect of test compounds on cellmetabolic activity, or for screening the effect of a geneticmanipulations on cells. The apparatus includes a multiwell devicedefining a plurality of discrete microwells on a substrate surface, at awell density of greater than about 100/cm², where the well volumes aresuch as to accommodate at most about 10⁶ cells/well, preferably between1-100 wells/cell, and structure for measuring the conductance in eachwell. The measuring structure includes (i) a pair of electrodes adaptedfor insertion into a well on the substrate, and (ii) circuitry forapplying a low-voltage, AC signal across the electrodes, when theelectrodes are submerged in the medium, and for synchronously measuringthe current across the electrodes, to monitor the level of growth ormetabolic activity of cells contained in the chamber.

In various preferred embodiments, the signal circuitry is effective togenerate a signal whose peak-to-peak voltage is between 5 and 10 mV, andincludes feedback means for adjusting the signal voltage level to aselected peak-to-peak voltage between 5 and 10 mV.

In other embodiments, the circuitry is designed to sample the voltage ofthe applied signal at a selected phase angle of the signal, oralternatively, to sample the voltage of the applied signal at afrequency which is at least an order of magnitude greater than that ofthe signal.

In another general aspect, the invention includes a high-throughputscreening method, e.g., for screening the effect of test compounds oncell metabolic activity, or the effect of a given genetic manipulation.The method includes placing cells in the wells of a multiwell devicedefining a plurality of discrete microwells on a substrate surface, at awell density of greater than about 100/cm², with the number of cells ineach well being less than about 10⁶, and preferably between 1-10³. Theconductance in each well is determined by applying a low-voltage, ACsignal across a pair of electrodes placed in that well, andsynchronously measuring the conductance across the electrodes.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a multiwell device that forms part ofthe apparatus of the invention;

FIG. 2A is an enlarged, fragmentary cross-sectional view of the devicein FIG. 1, with the wells in the device each containing a small numberof cells suspended in a culture medium;

FIG. 2B is a view like that in FIG. 2A, but further showing an electrodecover on the device;

FIG. 3 illustrates the grid of conductive wires in the electrode plateshown in FIG. 2B;

FIGS. 4A-4C illustrate another embodiment of the apparatus, heredesigned for well-by-well conductance measurements;

FIG. 5 shows idealized plots of conductance, as a function of time, inthe presence and absence of a library compound that inhibits cell growthor metabolism;

FIG. 6 is a block diagram depicting selected portions of the dataacquisition board and of the measurement input/output board inaccordance with the present invention; and

FIG. 7 depicts waveforms for the alternating current voltage suppliedfor application across a pair of pins, an output signal produced by acomparator in response to the alternating current voltage, and ahypothetical output signal from a sample-and-hold amplifier.

DETAILED DESCRIPTION OF THE INVENTION

A. Screening Apparatus and Method

FIG. 1 shows a perspective view of a multiwell device 8 formed of asubstrate 10 having a plurality of microwells, such as wells 12, on theupper substrate surface. In the embodiment shown, the microwells areformed by a grid of hydrophobic lines, such as lines 16 extendinglengthwise, and lines 18 extending widthwise. The lines are preferablyformed of a hydrophobic polymer material, such as polyethylene orpolystyrene, and are laid down in a conventional manner, e.g.,deposition of melted polymer from an applicator, or heat-mediatedattachment of a polymer grid fabric directly to the substrate surface.

Spacing between adjacent parallel lines is preferably 20-200 μm, so thatthe wells formed by intersecting lines have area dimensions of betweenabout 400 to 40,000 μm². The density of wells on the substrate is atleast 100/cm² and more preferably 10³/cm² to 10⁴/cm or greater.

The height (surface relief) of the grid lines, seen best in FIGS. 2B and2B, is typically between 20-200 μm. Microwell volumes, defined by thevolume that can be held as a discrete droplet in a microwell, aretypically in the range 10⁴ to 2 nanoliters.

The microwells in the device may be filled with selected biologicalcells by applying a suspension of the cells, at a desired cell density,over the device's surface, and allowing excess suspension fluid to drainoff, e.g., by blotting the edges of the device. This is illustrated inFIGS. 2A and 2B which show cell suspension droplets, such as droplet 20,in the microwells of the device, such as microwell 22. It will beappreciated that the droplet meniscus may extend above the height of thegrid lines.

The cell density is adjusted so that the wells are filled, on average,with a selected number of cells which is preferably between 1-100, butmay be as high as 10³ per well. As illustrated in FIGS. 2A and 2B, thedensity of the cells is such that the device has an average of about 2cells/well. A greater number of cells/well, e.g., an average of10-100/well provides improved statistical correlation among eventsobserved in different cells, due to more uniform cell-numberdistribution in the microwells of the device. At the same time, a smallnumber of cells, e.g., 10-100, allows a microwell (microvolume) formatin which desired concentrations of test compounds can be achieved withvery small amounts of compound, e.g., in the femptogram to nanogramrange. The actual number of cells employed will depend on the particulartype of cell and medium, and the confluency requirements of the cells.

With reference particularly to FIG. 2B, the apparatus of the invention,further includes means for measuring the conductance of the cell mediumin each well. In the embodiment shown, this means includes amulti-electrode cover 27 having a plurality of electrodes pairs, such aselectrode pair 26 made up of electrodes 26 a, 26 b formed as a grid onthe lower side of the cover. Specifically, the grid of electrode pairsmatches that of microwells in device 8, so that placement of the coveron the device places an electrode pair in each microwell.

The measuring means also includes a signal unit 28 electricallyconnected to the cover as described below with reference to FIG. 3. Theoperation of the unit to provide a low-voltage AC signal to eachelectrode, and interrogate the electrode to determine the conductance ineach microwell is described below with reference to FIGS. 6 and 7.

FIG. 3 is a plan view of cover 27, showing the grid pattern ofconductive wires, such as longitudinal wires 30, and lateral wires 32,connecting the electrode pairs in the cover. Each longitudinal wire isconnected to an electrode connector, such as wires 32 connected toconnectors 34, in a multi-connector array 36 along one side of thecover, as shown. Similarly, each lateral wire is connected to anelectrode connector, such as wires 32 connected to connectors 38, in amulti-connector array 40 along another side of the cover. The two arraysare designed to plug into matching ports in the signal unit.

As seen in FIG. 2B, each longitudinal wire, such as wire 30, iselectrically connected to one of the two electrodes in the longitudinalone-dimensional array of electrode pairs, such as the array includingpair 26, adjacent the wire. Similarly, each lateral wire, such as wire36, is electrically connected to one of the two electrodes in thelateral one-dimensional array of electrode pairs, such as the arrayincluding pair 26, adjacent that wire.

Thus, to interrogate a particular microwell in the device, the signalunit applies a low-voltage signal across the two connectors in cover 27which are connected to the two electrodes in that well. For example, tointerrogate microwell 26 in FIG. 2B, the signal unit applies a voltagesignal across connectors 34, 38 connected to electrodes 26 a, 26 b,respectively, forming the electrode pair in that microwell. To this end,the signal unit includes the basic electronics for applying alow-voltage signal to the electrodes, and for synchronously measuringthe current across the electrodes, as described below.

Unit 28 also includes conventional multiplexing or sampling circuitryfor alternately and successively interrogating each microwell, byapplying a short duration signal to successively to each well, andmeasuring the current across the “stimulated” electrodes in accordancewith the signalling and current measuring procedures described below.According to one feature of the signal unit, the time requiredaccurately interrogate each microwell can be quite short, on the orderof only a few cycles on the applied signal, allowing large arrays to becontinuously monitored in real time.

FIGS. 4A-4C illustrate a HTS apparatus 42 constructed according toanother embodiment of the invention. The apparatus includes a multiwelldevice 44 similar to above described device 8, and having a planar arrayof microwells, such as wells 46, 48. In this embodiment, however, themeasuring means for determining the conductance of each well is carriedout by an electrode arm 50 having a pair of electrodes 52, 54 adapted tobe received in a selected microwell of the device, and connected to asignal unit 56. The electrode arm is movable in the “z” plane betweenraised and lowered positions in which the electrodes are position above,and in a selected microwell, respectively, as illustrated in FIGS. 4Aand 4B, respectively. This movement is produced by a vertical actuator,indicated by arrow 58, which is also under the control of unit 50.

Also forming part of the apparatus is a stage 60 on which the device isplaced during operation. The stage is movable, in an “x-y” plane underthe control of signal unit 56, to alternatively and successively bringeach microwell in the device to an interrogation position directly belowthe electrode arm, as indicated for well 46 in FIGS. 4A and 4B. Thesignal unit also includes the basic electronics for applying alow-voltage signal to the electrodes, and for synchronously measuringthe current across the electrodes, as described below.

In an exemplary operation, for use in screening combinatorial librarycompounds, and with reference particularly to the embodiment shown inFIGS. 1-3, the microwells in the device are filled with a cellsuspension, as above, and the library compounds are added to each ofwells, either before or after cell addition. For example, usingmicrofabrication techniques of the type described in U.S. Pat. No.5,143,854, a position-addressable planar array of polymer librarymolecules is formed on a planar substrate in this case having orsubsequently prepared to have a hydrophobic grid forming the microwells,which correspond to the individual library-polymer regions. Afteraddition of the cells, the library compounds may be released forinteraction with the cells, e.g., by inclusion in the cell suspension ofan enzyme capable of cleaving the library molecules from the substratesurface.

Alternatively, the library compounds may be contained on a grid of pinsor the like corresponding to the microwell grid, allowing the compoundsto be simultaneously introduced into the wells, and released into thecorresponding wells, e.g., by enzymatic cleavage of a linker.

Alternatively, the library compounds may be distributed well-by-wellinto the microwell device, either as single compounds or mixtures oflibrary compounds.

After introducing the compound to be tested, the microwells areinterrogated to determine the conductance of medium in each cell, bymeasuring the current across the electrodes. In the present example, itis assumed that (i) a large number of individual library compounds areadded to the device, one per well, and (ii) one or more of the compoundsis able to inhibit metabolic activity and/or replication of the cells.In the absence of any inhibition, cell metabolism and growth will occurnormally, leading to an increase in measured conductance over time, asillustrated by plot number 1 in FIG. 5. Since only a few of the testcompounds will be expected to have an inhibitory effect, most or nearlyall of the plots will be represented by the “normal” plot.

Where an inhibitory compound is present, this will be evidenced by alower rate of conductance change over time, as indicated by curves 2 and3 in FIG. 5, where curve 2 represents moderate inhibition, and curve 3,nearly complete inhibition. The reduced conductance may be due toreduced metabolism and/or reduced replication. To confirm the latterpossibility, the microwells of interest may be further examined for cellcount, using standard cell counting methods.

In another general embodiment, the screening method is employed tomonitor the success of a selected genetic manipulation, e.g.,transformation of the cells with a selected vector, treatment with atransforming virus such as EBV, or cell fusion. In this embodiment, thegenetically manipulated cells are distributed on the microwell device asabove, and changes in cell conductance, related to cell replication aremonitored. Those wells that show significant increase in cellconductance over time are then selected as cells which are successfullymanipulated. Thus, for example, if the cells are transformed with avector containing a selectable antibiotic marker gene, cells which growin the presence of the antibiotic can be readily identified by theincreased conductance in the corresponding microwell(s).

B. Signal Unit Construction and Operation

FIG. 6 is a block diagram in accordance with one preferred embodiment ofthe present invention depicting a portion of the electronic circuit forapplying the potential across a pair of pins or electrodes 26 a, 26 b,and for monitoring the current across the pins.

A computer program executed by a computer system included in the cellculture monitoring and recording system causes a programmable gainamplifier 70 included in a data acquisition board 72 to transmit avoltage representative of that applied across a pair of pins 26 a, 26 binserted into a well for digitization by a measurement input/outputboard 74. The measurement input/output board 74 of the presentembodiment is preferably a MetraByte DAS-8 Data Acquisition and ControlBoard marketed by Keithley Metrabyte Corporation of Taunton, Mass.

To supply the alternating current voltage that is applied across aselected pair of pins 26 a, 26 b, the data acquisition board 72 includesa programmable voltage source 76. The programmable voltage source 76includes an alternating current generator 78 that produces a 370 Hz±20%, 10 volt peak-to-peak sine wave signal. The output signal producedby the alternating current generator 78 is transmitted to a programmableattenuator 80 also included in the programmable voltage source 76.Digital excitation level control signals supplied from the computersystem to the programmable attenuator 80 via excitation level controlsignal lines 82 permit adjustment of the peak-to-peak voltage suppliedto a first terminal 84 of a resistor, such as a 20.04 K ohm resistor 86.

A second terminal 88 of the resistor 86 connects to a bank of switches90. One of the switches 90 is selected by the computer system forapplying the alternating current voltage supplied by the programmablevoltage source 76 to a pair of pins 26 a, 26 b that extend into the wellbeing monitored.

The AC voltage applied across a pair of pins 26 a, 26 b is also suppliedto the input of the programmable gain amplifier 70. The gain of theamplifier 70 may be adjusted by control signals supplied from thecomputer system via gain control signal lines 92. The output signalsfrom the programmable attenuator 80, and from the programmable gainamplifier 70 are both supplied to a multiplexer 94. Control signalssupplied from the computer system to multiplexer 94 via multiplexercontrol signal lines 96 select one of these three signals forapplication to an input of a sample-and-hold amplifier 100 included inthe measurement input/output board 74.

The output signal from the sample-and-hold amplifier 100 is supplied tothe input of an analog-to-digital converter 102 also included in themeasurement input/output board 74. In addition to being supplied to theprogrammable attenuator 80, the 10 volt peak-to-peak output signal fromthe alternating current generator 78 is also supplied to the input of acomparator 104. The output signal from the comparator 104 changes stateeach time the alternating current voltage produced by the alternatingcurrent generator 78 passes through zero volts.

Thus, while the alternating current voltage produced by the generator 78has a potential greater than zero volts, the output signal from thecomparator 104 is in one state, and while that voltage has a potentialless than zero volts, the output signal from the comparator 104 is inits other state. The output signal from the comparator 104 is suppliedto a programmable timer 106 included in the measurement input/outputboard 74.

As described herein, the voltage present at the second terminal 88 ofresistor 86 is applied across the two pins of a selected well 12 via aswitch 90. This “pin voltage”, which is proportional to the conductivityof the medium and the current flow between the two pins, is measured bythe programmable gain amplifier 70. To efficiently obtain a reliablemeasurement of this voltage (and of the underlying current), the pinvoltage is preferably sampled synchronously with the applied voltage.This can be done in several ways, two of which are described below.

1. Sampling at a selected phase angle. FIG. 7 depicts a sinusoidalalternating current waveform 110 for the voltage present at the outputof the alternating current generator 78 together with the a digitalwaveform 112 of the output signal produced by the comparator 104. Duringinitialization of the cell culture monitoring and recording system andat any subsequent time that it is requested by an operator of the cellculture monitoring and recording system, the computer program executedby the computer system executes a procedure for establishing a delayperiod (“D”) of a selected duration that is shorter than one cycle ofthe sine waveform 110. For example, in FIG. 7, the delay period beginswhen the sine waveform 110 is changing from a positive potential to anegative potential has a potential of zero volts, and ends when the sinewaveform 110 has its immediately subsequent maximum positive value.

In measuring the delay period D, the computer program uses the outputsignal from the comparator 104 in the data acquisition board 72 togetherwith the programmable timer 106 included in the measurement input/outputboard 74 to determine the duration of one period of the sine waveform110. The computer program then establishes the delay period D atthree-fourths of one period of the sine waveform 110. Having determineda proper delay period D, the computer program then loads that delayperiod into the programmable timer 106 so that all subsequentmeasurements of the electrical potential across a pair of pins 26 a, 26b will occur when the voltage supplied to the first terminal 84 of theresistor 86 reaches its maximum value, i.e., at the same selected phaseangle of each cycle.

In measuring the voltage applied across a pair of pins 26 a, 26 b, theprogrammable timer 106 begins measuring each delay period at the instantat which the sine waveform 110 is changing from a positive potential toa negative potential has a potential of zero volts, i.e. immediatelyafter the digital value of the output signal from the comparator 104supplied to the programmable timer 106 changes from 0 to 1. When thedelay period D expires, the programmable timer 106 causes thesample-and-hold amplifier 100 to sample and hold the voltage of thesignal supplied from the output of the programmable gain amplifier 70via the multiplexer 94 as illustrated in the waveform 114 depicted inFIG. 7.

The programmable timer 106 also causes the analog-to-digital converter102 to convert the voltage of the analog signal received from thesample-and-hold amplifier 100 into a digital form. Subsequently, thisdigital number is transferred from the measurement input/output board 74to the computer system for storage as raw data suitable for subsequentanalysis and graphic display.

2. Sampling at a frequency which is at least an order of magnitudegreater than the applied voltage. Another way of synchronously samplingthe pin voltage, which is proportional to the current across theelectrodes, is to sample and digitize the signal at a frequency which isat least an order of magnitude greater than the applied voltage (termed“burst sampling”). In this mode of operation, the pin voltage is sampledand digitized a selected number of times (e.g., 10-1000) during a singlecycle of the applied voltage. This can be accomplished, for example, bytriggering the beginning and of storage of a string of digitized currentvalues with the rising or falling transition of the digital waveform 112of the output signal produced by the comparator 104. Such a digitizedwaveform can be analyzed with respect to the applied voltage using thecomputer system to calculate, for example, any phase lead or lag of theunderlying current with respect to the applied voltage, as well as thepeak-to-peak and/or RMS current values. These current values can in turnbe used in the calculation of the conductance of the medium as describedherein. An advantage of this approach is that an accurate estimate ofthe conductance can be obtained in a single cycle of the appliedvoltage, enabling rapid multiplex sampling of a plurality of samples.

Adjusting Voltage Applied Across a Pair of Pins. In addition toperforming the above, the program executed by the computer system alsodetermines the alternating current voltage to be applied from the secondterminal 88 of the resistor 86 across the pair of pins 26 a, 26 b duringsuch monitoring. To determine this alternating current voltage, thecomputer program first adjusts the programmable attenuator 80 so apotential of approximately 10 millivolts is present at its output and atthe first terminal 84 of the resistor 86. Because the 20.04 K ohmresistance of the resistor 86 separates its first terminal 84 from itssecond terminal 88, and because any cell growth media held in the well12 provides some electrical conductivity between the pair of pins 26 a,26 b inserted therein, initially the voltage at the second terminal 88and across a pair of pin 26 a, 26 b must be less than the value of 10millivolts intended to be used in measuring the conductivity between apair of pins 26 a, 26 b. The computer program then causes themultiplexer 94 to select the output signal from the programmable gainamplifier 70 for application to the input of the sample-and-holdamplifier 100, sets the gain of the programmable amplifier 70 so a peakvoltage of 10 millivolts at the second terminal 88 of the resistor 86will result in the analog-to-digital converter 102 producing a digitalnumber that is approximately 83.3% of the full range of theanalog-to-digital converter 102, and causes the bank of switches 90 toselect a pair of pins 26 a, 26 b for application of the alternatingcurrent voltage.

The cell culture monitoring and recording system then measures the peakalternating current voltage present at the second terminal 88 of theresistor 86 that is applied across the pair of pins 26 a, 26 b. If thevoltage at the second terminal 88 and across the pair of pins 26 a, 26 bis less than 5 millivolts, the computer program doubles the alternatingcurrent voltage produced by the programmable attenuator 80 repeatedlyuntil the voltage measured at the second terminal 88 exceeds 5millivolts. Having thus applied and measured an alternating currentvoltage across the pair of pins 26 a, 26 b that exceeds 5 millivolts andknowing the setting for the programmable attenuator 80 which producesthat voltage, the computer program then computes a new setting for theprogrammable attenuator 80 that will apply approximately a 10 millivoltalternating current voltage to the second terminal 88 and across thepair of pins 26 a, 26 b, and then transmits control signals setting theattenuator 80 to the computed value. Having established the value forthe alternating currant voltage applied by the programmable attenuator80 to the first terminal 84 of the resistor 86, the system is nowprepared to monitor and record the electrical conductivity of the well12. In measuring the conductivity of the well 12, the computer programfirst repetitively measures the voltage applied across the pair of pins26 a, 26 b and at the second terminal 88 of the resistor 86. Forexample, applying the method of measuring at a selected phase angle, thecomputer system collects 16 successive values for this voltage, and thecomputer program then computes an average of the 16 values using abox-car filter to obtain a single, average value for the voltage acrossthe pair of pins 26 a, 26 b. Alternatively, applying the burst samplingapproach, a single, average value can be obtained from the RMS value ofthe digitized signal. Using the single value of the pin voltage, thevalue of the voltage supplied by the programmable attenuator 80 to thefirst terminal 84 of the resistor 86, and the resistance of the resistor86; the computer program then computes the conductivity of the cellgrowth media and cells, if any, between the pair of pins 26 a, 26 b.

Having determined the conductivity between the pair of pins 26 a, 26 bfor this well 12, the computer program first stores the conductivityvalue for subsequent analysis and then proceeds to measure theconductivity between another pair of pins 26 a, 26 b extending intoanother well 12 in the microwell device 8. In determining theconductivity of each well, the cell culture monitoring and recordingsystem uses the procedures set forth above of first adjusting thealternating current voltage applied across the pair of pins 26 a, 26 b,and then measuring and averaging the voltage applied across the pair ofpins 26 a, 26 b extending into the well 12. This adjusting of theapplied voltage and determining of cell conductivity is repeated overand over until a conductivity has been determined and stored for allwells 12 in the microwell device 8.

At least one of the wells 12 in the microwell device 8 is preferably areference well that holds only cell culture media without cells.Furthermore, this reference well must be specifically so identified tothe analysis computer program because that program uses the conductivityvalue for the reference well in analyzing the conductivity for all theother wells.

Analysis of the conductivity of a well that held both cell growth mediaand cells included dividing the conductivity measured for the referencewell by the conductivity measured for the well that held both the cellgrowth media and cells. Rather than using the electrical conductivity ofthe reference well as the numerator of a fraction in analyzing theconductivity of a well that holds both cell growth media and cells, ithas been found more advantageous in analyzing the conductivity of wellsholding both cell growth media and cells to subtract the conductivitydetermined for the reference well from the conductivity determined forthe well holding both cell growth media and cells. Subtracting theconductivity measured for the reference well, i.e. a well that holdsonly cell growth media without cells, from the conductivity measured forwells that holds both growth media and cells removes the electricalconductivity of the cell growth media from the data for such wells.Removing the cell growth media conductivity from the data for the wellsresults in data values for the wells holding both cell growth media andcells that more closely represents the electrical conductivity of onlythe cells themselves, and the cells' metabolic products.

While the preferred embodiments of the present invention as describedabove employ a sinusoidal alternating current in monitoring cellcultures, it may be possible to employ any periodic voltage waveformthat is symmetric about zero volts in determining conductivity between apair of pins 26 a, 26 b. Thus, for example, a system for monitoring andrecording cell cultures in accordance with the present invention couldemploy an alternating current voltage having a triangular waveform.

Since a triangular waveform alternating current voltage may be easilygenerated using a digital logic circuit, in a system employing such awaveform it would be unnecessary to directly measure, as describedabove, the delay period D. Rather the digital circuits used ingenerating the triangular waveform alternating current voltage couldthemselves directly produce signals for controlling the operation of thesample-and-hold amplifier 100 and the analog-to-digital converter 102.However, such a system for monitoring and recording cell cultures wouldmerely employ a different, well known technique for determining thedelay period for its alternating current voltage that i is equal to aninterval of time between the alternating current voltage having aninstantaneous potential of zero volts and having an instantaneouspotential equal to the maximum voltage of the alternating currentvoltage.

Although the invention has been described with respect to particularembodiments and features, it will be appreciated that various changesand modifications can be made without departing from the invention. Asan example, and in another preferred embodiment, the probes are insertedinto the well from the bottom of the well to allow for easysterilization of the unit and minimal media volume.

What is claimed is:
 1. High throughput screening apparatus comprising asubstrate defining a plurality of discrete microwells on a substratesurface, at a well density of greater than about 100/cm², where the wellvolumes are such as to accommodate at most about 10³ cells/well, meansfor measuring the conductance in each microwell, said means including(i) a pair of electrodes adapted for insertion into a well on thesubstrate, and (ii) signal means for applying a low-voltage, AC signalacross said electrodes, when the electrodes are submerged in suchmedium, and for synchronously measuring the current across theelectrodes, to monitor the level of growth or metabolic activity ofcells contained in each well.
 2. The apparatus of claim 1, wherein saidsignal means is effective to generate a signal whose peak-to-peakvoltage is between 5 and 10 mV.
 3. The apparatus of claim 2, whereinsaid signal means includes feedback means for adjusting the signalvoltage level to a selected peak-to-peak voltage between 5 and 10 mV. 4.The apparatus of claim 1, wherein said signal means is designed tosample the voltage of the applied signal at a selected phase angle ofthe signal.
 5. The apparatus of claim 1, wherein said signal means isdesigned to sample the voltage of the applied signal at a frequencywhich is at least an order of magnitude greater than that of saidsignal.
 6. The apparatus of claim 1, wherein said wells are adapted tohold at most between 1-100 cells/well.
 7. High throughput screeningapparatus comprising: a) a substrate comprising a plurality of discretemicrowells on a substrate surface, at a well density of greater thanabout 100/cm², where the well volumes are such as to accommodate at mostabout 10³ cells/well; and b) a system comprising: i) at least one pairof electrodes adapted for insertion into a first well on the substrate;and (ii) circuitry adapted for applying a low-voltage, AC signal acrosssaid first pair of electrodes when the electrodes are submerged inmedium in said first well, and for synchronously measuring the currentacross the electrodes.
 8. The apparatus of claim 7, wherein saidcircuitry is effective to generate a signal whose peak-to-peak voltageis between 5 and 10 mV.
 9. The apparatus of claim 8, wherein saidcircuitry includes feedback system for adjusting the signal voltagelevel to a selected peak-to-peak voltage between 5 and 10 mV.
 10. Theapparatus of claim 7, wherein said circuitry is designed to sample thevoltage of the applied signal at a selected phase angle of the signal.11. The apparatus of claim 7, wherein said circuitry is designed tosample the voltage of the applied signal at a frequency which is atleast an order of magnitude greater than that of said signal.
 12. Theapparatus of claim 7, wherein said wells are adapted to hold at mostbetween 1-100 cells/well.